Human cytomegalovirus (CMV) replicates productively in cultured human fibroblasts, but most nonhuman cells or nonfibroblastic human cell cultures are nonpermissive and support only abortive infections. At least two different mechanisms can be postulated to explain the nonpermissive state: the absence of some essential cellular gene product(s) in nonpermissive cells, or the presence of some inhibitory product(s). We will evaluate these alternatives by examining CMV replication in: (1) hybrid cells derived from one permissive human parent and one nonpermissive mutant murine parent by polyethylene glycol-induced fusion and cloning in HAT or HAT-ouabain selective medium: (2) heterokaryons derives from human and murine parental cells prelabeled with different fluorescent probes (Hoechst 33258, R18, F18) and isolated immediately after fusion using a fluorescence-activated cell sorter (FACS); and (3) partial hybrids created by fusion between microcells carrying only small numbers of chromosomes and intact cells. Microcells are produced by cytochalasin B-induced enucleation of colchicine-treated cells. The murine-human hybrids will preferentially segregate human chromosomes, the heterokaryons will contain essentially complete sets of both parental genomes, and the partial hybrids, if derived from intact human cells and murine microcells, will be the equivalent of hybrids segregating murine chromosomes. All these cells will be screened for ability to sustain CMV replication using plaque assat, electron microscopy, density gradient centrifugation to detect viral DNA, and indirect immunofluorescence to detect viral antigen synthesis. We will determine from these criteria whether nonpermissiveness behaves as an expressed or repressed trait in each cross, and will correlate this with the chromosome composition of the cells as determined by banding techniques, in order to identify chromosomes essential for CMV expression or repression.